Use of cd36 to identify cancer subjects for treatment

ABSTRACT

Provided herein are methods for identifying a subject with cancer for treatment with a Psap peptides. The subject is identified based on a level of CD36. Also provided herein are compositions and methods for treatment of a subject with cancer based on a level of CD36.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.Provisional Application No. 61/782,850, filed Mar. 14, 2013, the entirecontents of which are incorporated by reference herein.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with U.S. Government support under R01CA135417awarded by the National Cancer Institute. The U.S. Government hascertain rights in the invention.

BACKGROUND OF INVENTION

Cancer remains a major public health priority. For example, an estimated7.6 million deaths from cancer occurred in 2008. Treatments for cancerare constantly improving as technology and science progresses.Unfortunately, it has become apparent that many cancer therapeutics areeffective only in subsets of cancer patients, even subsets of patientshaving the same type of cancer. As a result, it is becoming increasinglyimportant to find ways to identify patients that are likely to respondto treatment.

SUMMARY OF INVENTION

Aspects of the disclosure are based in part on the discovery thatelevated levels of CD36 in tumor cells indicate that a subject isresponsive to or is likely to be responsive to treatment with a Psappeptide. Accordingly, aspects of the disclosure relate to methods forevaluating a subject's responsiveness to treatment with a Psap peptideby determining a level of CD36 in a sample, such as a tumor sample. Insome embodiments, the methods described herein relate to identificationor selection of a subject for treatment with a Psap peptide based on alevel of CD36 in a sample. Other aspects of the disclosure relate tocompositions and methods for treatment of a subject with cancercharacterized by an elevated level of CD36.

In some aspects, the disclosure relates to a method for evaluating asubject's responsiveness to treatment with a Psap peptide, the methodcomprising determining a level of CD36 in a sample obtained from asubject having cancer, wherein an elevated level of CD36 in the samplecompared to a control level indicates that the subject is responsive toor likely to be responsive to treatment with a Psap peptide. In someembodiments, the level of CD36 in the sample is determined by performingan assay. In some embodiments, the method further comprises identifyingthe subject with an elevated level of CD36 in the sample compared to thecontrol level as responsive to or likely to be responsive to treatmentwith a Psap peptide. In some embodiments, the method further comprisesadministering to the subject identified as responsive to or likely to beresponsive to treatment with a Psap peptide an effective amount of aPsap peptide to treat the cancer.

Other aspects of the disclosure relate to a method for treating asubject with cancer, the method comprising administering to a subjectwith cancer characterized by an elevated level of CD36 in a samplecompared to a control level an effective amount of a Psap peptide totreat the cancer. In some embodiments, the control level is a level ofCD36 from a non-cancerous cell or tissue obtained from the subjecthaving the cancer. In some embodiments, the control level is a level ofCD36 in a cell or tissue obtained from a healthy subject or a populationof healthy subjects. In some embodiments, the control level is apredetermined level. In some embodiments, the level of CD36 is a CD36protein level.

Further aspects of the disclosure relate to a method for treating asubject with cancer, the method comprising (a) selecting a subject withcancer on the basis that the subject is known to have an elevated levelof CD36 in a sample compared to a control level; and (b) administeringan effective amount of a Psap peptide to the subject because the subjecthas an elevated level of CD36 in the sample compared to the controllevel. In some embodiments, the control level is a level of CD36 from anon-cancerous cell or tissue obtained from the subject having thecancer. In some embodiments, the control level is a level of CD36 in acell or tissue obtained from a healthy subject or a population ofhealthy subjects. In some embodiments, the control level is apredetermined level. In some embodiments, the level of CD36 is a CD36protein level.

In some embodiments of any of the methods provided herein, the cancer isprostate cancer, breast cancer, ovarian cancer, lung cancer, leukemia,pancreatic cancer, glioblastoma multiforme, astrocytoma, or melanoma.

In some embodiments of any of the methods provided herein, the Psappeptide comprises the amino acid sequence CDWLPK (SEQ ID NO: 1), DWLPK(SEQ ID NO: 2), or DWLP (SEQ ID NO: 3), or an amino acid substitutionvariant thereof, wherein the amino acid substitution is:

a) Tyrosine (Y) for Tryptophan (W);

b) an amino acid substitution for Leucine (L) selected from Valine (V),Alanine (A) or Glycine (G), or a non-canonical amino acid of similarsize, or a derivative thereof;

c) Arginine (R) for Lysine (K);

d) a D-isomer of Aspartic Acid (D) for an L-isomer of Aspartic Acid (D)and/or a D-isomer of Leucine (L) for a L-isomer of Leucine (L);

e) a D-isomer of Tryptophan (W) for an L-isomer of Tryptophan (W) and/ora D-isomer of Proline (P) for an L-isomer of Proline (P); orcombinations thereof. In some embodiments, the Psap peptide is 50 aminoacids or fewer in length. In some embodiments, the Psap peptide is 30amino acids or fewer in length. In some embodiments, the Psap peptide is15 amino acids or fewer in length. In some embodiments, the Psap peptideis 6 amino acids or fewer in length. In some embodiments, the Psappeptide is a cyclic peptide. In some embodiments, the non-canonicalamino acid of similar size is methylvaline, methylleucine, or sarcosine.

In yet another aspect, the disclosure relates to a composition for usein treating a subject with cancer characterized by an elevated level ofCD36 in a sample compared to a control level, the composition comprisinga Psap peptide.

In another aspect, the disclosure relates to use of a composition for inthe manufacture of a medicament for treating a subject with cancercharacterized by an elevated level of CD36 in a sample compared to acontrol level, the composition comprising a Psap peptide.

In some embodiments of a use or composition provided herein, the controllevel is a level of CD36 from a non-cancerous cell or tissue obtainedfrom the subject having cancer. In some embodiments of a use orcomposition provided herein, the control level is a level of CD36 in acell or tissue obtained from a healthy subject or a population ofhealthy subjects. In some embodiments of a use or composition providedherein, the control level is a predetermined level. In some embodimentsof a use or composition provided herein, the level of CD36 is a CD36protein level.

In some embodiments of a use or composition described herein, the canceris prostate cancer, breast cancer, ovarian cancer, lung cancer,leukemia, pancreatic cancer, glioblastoma multiforme, astrocytoma, ormelanoma.

In some embodiments of a use or composition described herein, the Psappeptide comprises the amino acid sequence CDWLPK (SEQ ID NO: 1), DWLPK(SEQ ID NO: 2), or DWLP (SEQ ID NO: 3), or an amino acid substitutionvariant thereof, wherein the amino acid substitution is:

a) Tyrosine (Y) for Tryptophan (W);

b) an amino acid substitution for Leucine (L) selected from Valine (V),Alanine (A) or Glycine (G), or a non-canonical amino acid of similarsize, or a derivative thereof;

c) Arginine (R) for Lysine (K);

d) a D-isomer of Aspartic Acid (D) for an L-isomer of Aspartic Acid (D)and/or a D-isomer of Leucine (L) for a L-isomer of Leucine (L);

e) a D-isomer of Tryptophan (W) for an L-isomer of Tryptophan (W) and/ora D-isomer of Proline (P) for an L-isomer of Proline (P); orcombinations thereof. In some embodiments, the Psap peptide is 50 aminoacids or fewer in length. In some embodiments, the Psap peptide is 30amino acids or fewer in length. In some embodiments, the Psap peptide is15 amino acids or fewer in length. In some embodiments, the Psap peptideis 6 amino acids or fewer in length. In some embodiments, the Psappeptide is a cyclic peptide. In some embodiments, the non-canonicalamino acid of similar size is methylvaline, methylleucine, or sarcosine.

In some embodiments of a method, composition or use provided herein, thesample is a tumor sample.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a graph showing proliferation of LLC cells 48 hours afteradditional of serially diluted amounts of recombinant Tsp-1 or DWLPK(SEQ ID NO: 2) peptide.

FIG. 1B is a photograph of a western blot showing that CD36 protein isexpressed in LLC cells.

FIG. 2 is a photograph of a western blot showing that CD36 protein isexpressed in breast cancer (MDA-231, MCF-7), ovarian cancer (ID8),melanoma (B16), prostate cancer (PC3 and LNCaP), and lung cancer (LLC)cell lines.

FIG. 3 is a photograph of a western blot showing that CD36 protein isexpressed in primary ovarian cancer cell derived from patient ascites.

FIG. 4 is a photograph of a western blot showing that CD36 protein isexpressed in pancreatic (AsPC1), ovarian (DF-14 and ID-8), breast(MDA-MB231 and LM2), prostate (PC3, PC3-M-LN4, LN-CAP, and LN-CAP-LN3),melanoma (B16-B16), and lung cancer (LLC) cells. Exemplary high and lowCD36 expressing cells lines are shown in boxes.

FIG. 5 is a graph showing that dW1P (SEQ ID NO: 47) peptide causedregression of cancer in a cancer model that expresses high levels ofCD36.

FIG. 6 is a graph showing that ovarian Cancer cells expressing CD36 aresensitive to Tsp-1 mediated cell killing.

FIG. 7 is a graph showing the primary tumor mass of mice injected withAsPC pancreatic cancer cells that express high levels of CD36 and thentreated with dW1P (SEQ ID NO: 47) peptide or control. The primary tumormass was inhibited by peptide treatment.

FIG. 8 is a graph showing that treatment of mice with B16-B16 melanomatumors (which express low levels of CD36) with dW1P (SEQ ID NO: 47)peptide inhibited tumor growth but did not regress the tumor.

DETAILED DESCRIPTION OF INVENTION

Psap peptides are therapeutic peptides containing amino acid sequencesthat were originally derived from fragments of Saposin A, a knownanti-angiogenic protein. Psap peptides generally comprise a coresequence of CDWLPK (SEQ ID NO: 1), DWLPK (SEQ ID NO: 2), or DWLP (SEQ IDNO: 3) or an amino substitution variant thereof and can be of a lengthof as few as 4 amino acids (e.g., a peptide that consists of DWLP (SEQID NO: 3) or an amino acid substitution variant). Such Psap peptideshave been shown previously to be effective for treating multiple typesof cancers (see, e.g., PCT publications WO2009002931 and WO/2011/084685;PCT application PCT/US2012/71424, published as PCT publicationWO/2013/096868, and U.S. patent application Ser. Nos. 12/640,788 and13/516,511, all of which are incorporated herein by reference in theirentirety). Administration of a Psap peptide was previously thought tostimulate thrombospondin (Tsp-1) in vivo, which in turn acted onendothelial cells causing an anti-angiogenic effect that resulted inindirect inhibition of cancer and/or metastatic growth.

As described herein, it has been discovered that tumor cells fromseveral different types of cancers that are responsive Psap peptidesexpress CD36. CD36 is a member of the class B scavenger receptor familyof cell surface proteins and has many ligands including oxidized lowdensity lipoprotein, oxidized phospholipids, long-chain fatty acids,collagen, and Tsp-1. Without wishing to be bound by any theory ormechanism, it is believed that administration of Psap peptide stimulatesTsp-1, which then acts directly on tumor cells by interacting with CD36on the tumor cells. The interaction between Tsp-1 and CD36 on the tumorcells may result in inhibition of tumor cell proliferation and/orinduction of tumor cell apoptosis. Thus, Psap peptides appear to treatcancer through two different independent mechanisms, indirectly throughan anti-angiogenic affect and directly by interaction of Tsp-1 with CD36on tumor cells. Thus, responsiveness of a subject with cancer totreatment with a Psap peptide may depend on the level of CD36 expressedby the cancer.

Accordingly, aspects of the disclosure relate to methods for evaluatinga subject's responsiveness to treatment with a Psap peptide bydetermining a level of CD36 in a sample, such as a tumor sample. In someembodiments, the methods described herein relate to identification orselection of a subject for treatment with a Psap peptide based on alevel of CD36 in a sample, such as a tumor sample. Other aspects of thedisclosure relate to compositions and methods for treatment of a subjectwith cancer characterized by an elevated level of CD36 (e.g., selectedor identified on the basis that the cancer has an elevated level of CD36in a sample compared to a control level).

As used herein, “responsive to treatment with a Psap peptide” includes,but is not limited to, prevention or reduction of the development of acancer, reduction of the symptoms of cancer, suppression or inhibitionof the growth of a cancer, prevention of metastasis and/or invasion ofan existing cancer, promotion or induction of regression of the cancer,inhibition or suppression of the proliferation of cancerous cells,reduction of angiogenesis and/or an increase in the amount of apoptoticcancer cells in response to treatment with a Psap peptide.

As used herein, “non-responsive to treatment with a Psap peptide”includes, but is not limited to, an absence of prevention or reductionof the development of a cancer, an absence of reduction of the symptomsof cancer, an absence of suppression or inhibition of the growth of acancer, an absence of prevention of metastasis and/or invasion of anexisting cancer, an absence of promotion or induction of regression ofthe cancer, an absence of inhibition or suppression of the proliferationof cancerous cells, an absence of reduction of angiogenesis and/or adecrease in the amount of apoptotic cancer cells in response totreatment with a Psap peptide.

Diagnostic and Theranostic Methods

Aspects of the disclosure relate to diagnostic and theranostic methodsuseful for evaluating a subject's responsiveness to treatment with aPsap peptide. In some embodiments, the method comprises determining alevel of CD36 in a sample obtained from a subject having cancer, whereinan elevated level of CD36 in the sample compared to a control levelindicates that the subject is responsive to or likely to be responsiveto treatment with a Psap peptide (i.e., if the level of CD36 in thesample is elevated compared to a control level, the subject isidentified as responsive or likely to be responsive to treatment with aPsap peptide). In some embodiments, the method further comprisesidentifying the subject with an elevated level of CD36 in the samplecompared to the control level as responsive to or likely to beresponsive to treatment with a Psap peptide. In some embodiments, themethod further comprises administering to the subject identified asresponsive to or likely to be responsive to treatment with a Psappeptide an effective amount of a Psap peptide described herein to treatthe cancer. In some embodiments, the sample obtained from a subjecthaving cancer is a tumor sample.

In some embodiments, an elevated level of CD36 in the sample compared toa control level indicates that the cancer will regress or is likely toregress in response to treatment with a Psap peptide. In someembodiments, the method further comprises identifying the subject withan elevated level of CD36 in the sample compared to the control level ashaving a cancer that will regress or is likely to regress in response totreatment with a Psap peptide. In some embodiments, the method furthercomprises administering to the subject identified having a cancer thatwill regress or is likely to regress in response to treatment with aPsap peptide an effective amount of a Psap peptide described herein tocause regression of the cancer.

As used herein, “an elevated level of CD36” means that the level of CD36is above a control level, such as a pre-determined threshold or a levelof CD36 in a control sample. Control levels are described in detailherein. An elevated level of CD36 includes a CD36 level that is, forexample, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,150%, 200%, 300%, 400%, 500% or more above a control level. An elevatedlevel of CD36 also includes increasing a phenomenon from a zero state(e.g., no or undetectable CD36 expression in a control) to a non-zerostate (e.g., some CD36 expression or detectable CD36 expression in asample).

As used herein, “treatment with a Psap peptide” is meant to compriseadministration of a Psap peptide to a subject. Psap peptides aredescribed herein. It is to be understood that treatment with a Psappeptide may include treatment with only a Psap peptide or may includetreatment with multiple agents or therapies, such as a Psap peptide andanother chemotherapeutic agent and/or another form of therapy such assurgery, radiotherapy, or chemotherapy.

Treatment

Other aspects of the disclosure relate to methods for treating a subjectwith cancer. In some embodiments, the method comprises administering toa subject with cancer characterized by an elevated level of CD36 in asample obtained from the subject compared to a control level aneffective amount of a Psap peptide described herein to treat the cancer.In some embodiments, the method comprises:

(a) selecting a subject with cancer on the basis that the subject isknown to have an elevated level of CD36 in a sample compared to acontrol level; and

(b) administering an effective amount of a Psap peptide to the subjectbecause the subject has an elevated level of CD36 in the sample comparedto the control level.

Other aspects of the disclosure relate to compositions and uses ofcompositions in the manufacture of a medicament for treating a subjectwith cancer characterized by an elevated level of CD36 in a sample. Insome embodiments, the composition comprises a Psap peptide as describedherein. In some embodiments, the sample is a tumor sample.

As used herein, “treat” or “treatment” includes, but is not limited to,preventing or reducing the development of a cancer, reducing thesymptoms of cancer, suppressing or inhibiting the growth of a cancer,preventing metastasis and/or invasion of an existing cancer, promotingor inducing regression of the cancer, inhibiting or suppressing theproliferation of cancerous cells, reducing angiogenesis and/orincreasing the amount of apoptotic cancer cells. In some embodiments,treatment of cancer is a direct inhibition or suppression of theproliferation of cancer cells and does not involve an inhibition orsuppression of angiogenesis (which indirectly leads to inhibition orsuppression of the proliferation of cancer cells).

An effective amount is a dosage of the Psap peptide sufficient toprovide a medically desirable result, such as treatment of cancer. Theeffective amount will vary with the particular cancer being treated, theage and physical condition of the subject being treated, the severity ofthe condition, the duration of the treatment, the nature of anyconcurrent therapy, the specific route of administration and the likefactors within the knowledge and expertise of the health practitioner.For administration to a subject such as a human, a dosage of from about0.001, 0.01, 0.1, or 1 mg/kg up to 50, 100, 150, or 500 mg/kg or morecan typically be employed.

Psap peptides and compositions thereof can be formulated for a varietyof modes of administration, including systemic, topical or localizedadministration. Techniques and formulations generally can be found inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,latest edition. When administered, a Psap peptide may be applied inpharmaceutically-acceptable amounts and in pharmaceutically-acceptablecompositions. Such preparations may routinely contain salt, bufferingagents, preservatives, compatible carriers, and optionally othertherapeutic agents. When used in medicine, the salts should bepharmaceutically acceptable, but non-pharmaceutically acceptable saltsmay conveniently be used to prepare pharmaceutically-acceptable saltsthereof and are not excluded from the scope of the disclosure. Suchpharmacologically and pharmaceutically-acceptable salts include, but arenot limited to, those prepared from the following acids: hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic,citric, formic, malonic, succinic, and the like. Also,pharmaceutically-acceptable salts can be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium or calcium salts.

A Psap peptide may be combined, optionally, with apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid filler, diluents or encapsulating substanceswhich are suitable for administration into a human. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the molecules of the present disclosure, and witheach other, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficacy. Some examplesof materials which can serve as pharmaceutically-acceptable carriersinclude: (1) sugars, such as lactose, glucose and sucrose; (2) starches,such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, methylcellulose,ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, suchas magnesium stearate, sodium lauryl sulfate and talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. The term “unit dose” when used in reference to apharmaceutical composition of the present disclosure refers tophysically discrete units suitable as unitary dosage for the subject,each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect in association withthe required diluent; i.e., carrier, or vehicle.

A variety of administration routes are available. The particular modeselected will depend upon the type of cancer being treated and thedosage required for therapeutic efficacy. The methods of the disclosure,generally speaking, may be practiced using any mode of administrationthat is medically acceptable, meaning any mode that produces effectivelevels of the active compounds without causing clinically unacceptableadverse effects. Such modes of administration include oral, rectal,topical, nasal, interdermal, or parenteral routes. The term “parenteral”includes subcutaneous, intravenous, intramuscular, or infusion.

In some embodiments, administration is parenteral. Injectablepreparations suitable for parenteral administration include, forexample, sterile injectable aqueous or oleaginous suspensions and may beformulated according to the known art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution, suspension or emulsion in anontoxic parenterally acceptable diluent or solvent, for example, as asolution in 1,3 propanediol or 1,3 butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,U.S.P. and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose any bland fixed oil may be employed includingsynthetic mono or di glycerides. In addition, fatty acids such as oleicacid find use in the preparation of injectables. The injectableformulations can be sterilized, for example, by filtration through abacterial-retaining filter, or by incorporating sterilizing agents inthe form of sterile solid compositions which can be dissolved ordispersed in sterile water or other sterile injectable medium prior touse.

For topical administration, the pharmaceutical composition can beformulated into ointments, salves, gels, or creams, as is generallyknown in the art. Topical administration can utilize transdermaldelivery systems well known in the art. An example is a dermal patch.Alternatively the biolistic gene gun method of delivery can be used. Thegene gun is a device for injecting cells with genetic information,originally designed for plant transformation. The payload is anelemental particle of a heavy metal coated with plasmid DNA. Thistechnique is often simply referred to as biolistics. Another instrumentthat uses biolistics technology is the PDS-1000/He particle deliverysystem. The composition described herein can be coated on minute goldparticles, and these coated particles are “shot” into biological tissuessuch as hemangiomas and melanoma under high pressure. An example of genegun-based method is described for DNA based vaccination of cattle byLoehr B. I. et al., J. Virol. 2000, 74:6077-86.

The pharmaceutical compositions described herein are also suitablyadministered by intratumoral, peritumoral, intralesional or perilesionalroutes, to exert local as well as systemic effects. The intraperitonealroute is expected to be particularly useful, for example, in thetreatment of ovarian tumors. For these uses, additional conventionalpharmaceutical preparations such as tablets, granules, powders,capsules, and sprays can be preferentially required. In suchformulations further conventional additives such as binding-agents,wetting agents, propellants, lubricants, and stabilizers can also berequired.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the anti-inflammatory agent. Other compositionsinclude suspensions in aqueous liquids or non-aqueous liquids such as asyrup, elixir or an emulsion.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the anti-inflammatory agent, increasing convenienceto the subject and the physician. Many types of release delivery systemsare available and known to those of ordinary skill in the art. Theyinclude polymer base systems such as poly(lactide-glycolide),copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters,polyhydroxybutyric acid, and polyanhydrides. Microcapsules of theforegoing polymers containing drugs are described in, for example, U.S.Pat. No. 5,075,109. Delivery systems also include non-polymer systemsthat are: lipids including sterols such as cholesterol, cholesterolesters and fatty acids or neutral fats such as mono- di- andtri-glycerides; hydrogel release systems; sylastic systems; peptidebased systems; wax coatings; compressed tablets using conventionalbinders and excipients; partially fused implants; and the like. Specificexamples include, but are not limited to: (a) erosional systems in whichthe anti-inflammatory agent is contained in a form within a matrix suchas those described in U.S. Pat. Nos. 4,452,775, 4,667,014, 4,748,034 and5,239,660 and (b) diffusional systems in which an active componentpermeates at a controlled rate from a polymer such as described in U.S.Pat. Nos. 3,832,253, and 3,854,480. In addition, pump-based hardwaredelivery systems can be used, some of which are adapted forimplantation.

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions. Long-term release, areused herein, means that the implant is constructed and arranged todelivery therapeutic levels of the active ingredient for at least 30days, and preferably 60 days. Long-term sustained release implants arewell-known to those of ordinary skill in the art and include some of therelease systems described above.

In some embodiments, the pharmaceutical compositions used fortherapeutic administration must be sterile. Sterility is readilyaccomplished by filtration through sterile filtration membranes (e.g.,0.2 micron membranes). Alternatively, preservatives can be used toprevent the growth or action of microorganisms. Various preservativesare well known and include, for example, phenol and ascorbic acid. Theactive ingredients and/or the pharmaceutical compositions ordinarilywill be stored in lyophilized form or as an aqueous solution if it ishighly stable to thermal and oxidative denaturation. The pH of thepreparations typically will be about from 6 to 8, although higher orlower pH values can also be appropriate in certain instances.

In some embodiments, administration of a Psap peptide may be combinedwith another therapy, such as a chemotherapy, radiation, and/or surgery.

CD36

CD36 (Cluster of Differentiation 36) is an integral membrane proteinfound on the surface of many cell types in vertebrate animals and isalso known as FAT, GP4, GP3B, GPIV, CHDS7, PASIV, SCARB3, and BDPLT10.The Entrez Gene ID for human CD36 is 948. Exemplary human CD36transcripts and proteins are below:

CD36 Transcript Variant 1 (SEQ ID NO: 4)CTTTCAATTCCTCTGGCAACAAACCACACACTGGGATCTGACACTGTAGAGTGCTTTCTCTTCTCTTTTTTTGGGGGGGGGAGGGGGTGTGGTTGCATATTTAAACTCTCACGCATTTATGTACTGAGGACTGCAGTGTAGGACTTTCCTGCAGAATACCATTTGATCCTATTAAGAATTGTCCAAATGTTGGAGCATTTGATTGAAAAATCCTTCTTAGCCATTTTAAAGATAGCTTTCCAATGATTAGACGAATTGATTCTTTCTGTGACTCATCAGTTCATTTCCTGTAAAATTCATGTCTTGCTGTTGATTTGTGAATAAGAACCAGAGCTTGTAGAAACCACTTTAATCATATCCAGGAGTTTGCAAGAAACAGGTGCTTAACACTAATTCACCTCCTGAACAAGAAAAATGGGCTGTGACCGGAACTGTGGGCTCATCGCTGGGGCTGTCATTGGTGCTGTCCTGGCTGTGTTTGGAGGTATTCTAATGCCAGTTGGAGACCTGCTTATCCAGAAGACAATTAAAAAGCAAGTTGTCCTCGAAGAAGGTACAATTGCTTTTAAAAATTGGGTTAAAACAGGCACAGAAGTTTACAGACAGTTTTGGATCTTTGATGTGCAAAATCCACAGGAAGTGATGATGAACAGCAGCAACATTCAAGTTAAGCAAAGAGGTCCTTATACGTACAGAGTTCGTTTTCTAGCCAAGGAAAATGTAACCCAGGACGCTGAGGACAACACAGTCTCTTTCCTGCAGCCCAATGGTGCCATCTTCGAACCTTCACTATCAGTTGGAACAGAGGCTGACAACTTCACAGTTCTCAATCTGGCTGTGGCAGCTGCATCCCATATCTATCAAAATCAATTTGTTCAAATGATCCTCAATTCACTTATTAACAAGTCAAAATCTTCTATGTTCCAAGTCAGAACTTTGAGAGAACTGTTATGGGGCTATAGGGATCCATTTTTGAGTTTGGTTCCGTACCCTGTTACTACCACAGTTGGTCTGTTTTATCCTTACAACAATACTGCAGATGGAGTTTATAAAGTTTTCAATGGAAAAGATAACATAAGTAAAGTTGCCATAATCGACACATATAAAGGTAAAAGGAATCTGTCCTATTGGGAAAGTCACTGCGACATGATTAATGGTACAGATGCAGCCTCATTTCCACCTTTTGTTGAGAAAAGCCAGGTATTGCAGTTCTTTTCTTCTGATATTTGCAGGTCAATCTATGCTGTATTTGAATCCGACGTTAATCTGAAAGGAATCCCTGTGTATAGATTTGTTCTTCCATCCAAGGCCTTTGCCTCTCCAGTTGAAAACCCAGACAACTATTGTTTCTGCACAGAAAAAATTATCTCAAAAAATTGTACATCATATGGTGTGCTAGACATCAGCAAATGCAAAGAAGGGAGACCTGTGTACATTTCACTTCCTCATTTTCTGTATGCAAGTCCTGATGTTTCAGAACCTATTGATGGATTAAACCCAAATGAAGAAGAACATAGGACATACTTGGATATTGAACCTATAACTGGATTCACTTTACAATTTGCAAAACGGCTGCAGGTCAACCTATTGGTCAAGCCATCAGAAAAAATTCAAGTATTAAAGAATCTGAAGAGGAACTATATTGTGCCTATTCTTTGGCTTAATGAGACTGGGACCATTGGTGATGAGAAGGCAAACATGTTCAGAAGTCAAGTAACTGGAAAAATAAACCTCCTTGGCCTGATAGAAATGATCTTACTCAGTGTTGGTGTGGTGATGTTTGTTGCTTTTATGATTTCATATTGTGCATGCAGATCGAAAACAATAAAATAAACCTGGCTCAAGCACAAACCAATTTGTGTTGTTCTGATTCAATAATTGGTTTCTGGGTGGCCAATTCAGAAGAAGAGTGTACATGCTCAACAAATCCTAGGCCCTGCATTCCTGTCATCCTCATCCGGGGGAAACACCATCATCCCAGTAGCTGCCCTATTCAACTGCAACAGTCTCCAGGACCATCAGTATACTGCATTTCATGTGCACCAAATATTTTGAAAGACATTTATAAATAATTGGCTTATGACTCATATTTCTCTATGAATACCTTCATACAGCAGGTATAACTCTTTTCTTTATGGGCTTAAATATTTTGTCACTGATCCTGCAAATGGACATCATTTTAGCACACTAGCGGTTTATATTTTAAGGACCTTCATTCTCTGTTCTGCACCTCTTCTGGAAATTGAGTAAATTTTGCTTTTTTTTTTTTACTCAGTTGCAACTTACGCTTGGCATCTTCAGAATGCTTTTCTAGCATTAAGAGATGTAAATGATAAAGGAATTATTGTATGAAATATTACAAAGCGTAGACTATGCATTGTTATTCATTATAATATTTTTTGCTGTCATAATCGCCTCATAAAGACAGGTTTCAACCATTAAAATATGTTCTTCCTTAAATTCCTGTGCTTTTTCTAGTTCCTCTTGTGTCATAAAATGTTTATCCTAATTTTCTCTCTGAAGTATATTTTATCTGAATCCACATTTCTTTATAAATCCATAGTCCTTGCTGAAATATGCTTTCTAAATTTCTACCACTTTGTTCTAGGCTAATTTTTTAAGCTAATTGGATGAAGAACAAAAAGACATTTGGTTTCATCCTTTACAGCAGTAGGACAATTGCAAAGGTTTTTCCTTTTTCATAAGGAGACACATTAATAGGTAACTCTGTTTCTTGAGCAGGGGTTCACTTATTCTGAGAGCATTAGTTCTCCTAAAAAGCTCCAGCATAGAAAGGGAAGATAAACCAAATTCTAGCTTGTGTTTTACCCACAGAAGGATACAGGACAAAGGAATAGTAACTGGCCTGTTTGGATACTAAAATCGAAAATAACTTTTAGCCTCCTCCTTATGATAGCCGCCAGAGTAAATGTTGAGCATTACTACAGAAAAGCCACAAACCAAGAATCTACCTGTTTGGAAAGATCTTTTGCATCTCTGAAGGTGCTTAAAGCATACTTAGTGCCTTTCCTTTTAACTGGGAAGATAAAAGAAGTATCTGTCCAAGATATTAATATGTAAGATAACATTGTAGACATGTTCTTCTGATAATACAAGGTTTATTCTATTTGCATTAGGATATTTGTGGACATGTCCATCTAATATAAAGGAAAGTTTTTTAATCATTGAGGCATGTAGGGCTGAGTTATATAATGTAGAAACTTCTAAAGATAATTGGATGAGAATATACATATTGACCTGTATATTATGACTAATCATGACTCAGATCTTAATACAGGGATGATCTCATAGCATTTAGATATCAGAAAAGGTTTTGACCTATATGTCTTTAATATTGTTTGAATACATGTATAATCTTTATCATTCCTCAGTGTTTCATTTCTCAAATTCTGTAAAAGGAATATAAGAGGAAAGACAATTCATATACAAAGACAACGAGATTAAAAATATGCAGTAGGAAAAATAATTACTTAAGGGGAGATTTTTTTTACATGAAATCTGGGCTTTGGATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCACATATGCACTGTGGTGGGAGTGGGGCAACTTGGGGAATATGTTACATGTGTGACTTTGTTTTGCCCTGGCGAAGTTAATGTTGTTCAGAAAGGGTAAATGTTTGGACACTTGCAATTGCTCATGGATGAATTTATATGTTTTAGTCATAGAAAAATTGTACCCTTTGATAGAAGCACATTTTCTTTCCAAAGCTGGTTATTAACCACAGAATTATAGCAGGTATTCATAACTTAAGTTTGAAAATCAATAGCGTCTGCAAATGGATTAACAGATTAGAGAATCAACAGCATCGGAAAATAGGTTAATGCATATTGCTTCTAACAAGTGCATGAAGAAATAGAAGAAGCTATGTAGCTTTCAGTTCTGACAGAAAAGGGTGAAGGAGGGTATCATTTCAAGAAAAAAAATAGCTATCACGCAATGGTTATCTCTGAAAATATTTGTATTAAGATGTGTATACATGGCCAGGCATGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCAGGTGGATCACGAGGTCAGGAGATCAAGACCATCCTGGCCAACATGGTGAAACCTCATCTCTACTAAAAATACAAAAATGAGCGGGGTGTGGTGGCCCATGCCTGTAGTCCCAGCTGCTCGGGAGACTGAATCTCTTGAGCCTGGGAAGCAGAGGTTGCAGTGAACTGAGATCGCGTCACTGCACTCCAGCCTGGTGACAGAGCGAGATTCCATCTCAAAAAAAAAAAACAGTATGCACGTACAAATTTCTTAACCTGTTATCAATGTCTGAGCTACATAATTATCTTTCTAGTTGGAGTTTGTTTTAGGTGTGTACCAACTGACATTTCAGTTTTTCTGTTTGAAGTCCAATGTATTAGTGACTCTGTGGCTGCTCTCTTCACCTGCCCCTTGTGGCCTGTCTACAATTCTAAATGGATTTTGAACTCAATGTCGTCGCTTCTGGTTTCCTGCATATACCAATAGCATTACCTATGACTTTTTTTTTCCTGAGCTATTTTCACTGAGCTGAGCTAATGAACTAAAACTGAGTTATGTTTAATATTTGTATCAAATACATAAAAGGAATACTGCTTTTTCCTTTTGTGGCTCAAAGGTAGCTGCATTTTAAAATATTTGTGAAAATAAAAACTTTTGTTATTAGAAAAATGA  CD36 Transcript Variant 2(SEQ ID NO: 5)GAGGATGTCAATGGCTTTCAGATGTCAGGATAACCTTAAGGATAGATGAAGGGTTGAGAGCCTGTGCCTCATTTCTGAGTTCTCAGCTGCTATGCCGTGGAAATCCTGTTTACTTTCTGCATCTGCTCCTGCAAGACTCTGGAGCCAGTCTTGAGGTCCTACATCTCCGAAAGCAAGCTCTTCTAGAAGTTGATAGCTTTCCAATGATTAGACGAATTGATTCTTTCTGTGACTCATCAGTTCATTTCCTGTAAAATTCATGTCTTGCTGTTGATTTGTGAATAAGAACCAGAGCTTGTAGAAACCACTTTAATCATATCCAGGAGTTTGCAAGAAACAGGTGCTTAACACTAATTCACCTCCTGAACAAGAAAAATGGGCTGTGACCGGAACTGTGGGCTCATCGCTGGGGCTGTCATTGGTGCTGTCCTGGCTGTGTTTGGAGGTATTCTAATGCCAGTTGGAGACCTGCTTATCCAGAAGACAATTAAAAAGCAAGTTGTCCTCGAAGAAGGTACAATTGCTTTTAAAAATTGGGTTAAAACAGGCACAGAAGTTTACAGACAGTTTTGGATCTTTGATGTGCAAAATCCACAGGAAGTGATGATGAACAGCAGCAACATTCAAGTTAAGCAAAGAGGTCCTTATACGTACAGAGTTCGTTTTCTAGCCAAGGAAAATGTAACCCAGGACGCTGAGGACAACACAGTCTCTTTCCTGCAGCCCAATGGTGCCATCTTCGAACCTTCACTATCAGTTGGAACAGAGGCTGACAACTTCACAGTTCTCAATCTGGCTGTGGCAGCTGCATCCCATATCTATCAAAATCAATTTGTTCAAATGATCCTCAATTCACTTATTAACAAGTCAAAATCTTCTATGTTCCAAGTCAGAACTTTGAGAGAACTGTTATGGGGCTATAGGGATCCATTTTTGAGTTTGGTTCCGTACCCTGTTACTACCACAGTTGGTCTGTTTTATCCTTACAACAATACTGCAGATGGAGTTTATAAAGTTTTCAATGGAAAAGATAACATAAGTAAAGTTGCCATAATCGACACATATAAAGGTAAAAGGAATCTGTCCTATTGGGAAAGTCACTGCGACATGATTAATGGTACAGATGCAGCCTCATTTCCACCTTTTGTTGAGAAAAGCCAGGTATTGCAGTTCTTTTCTTCTGATATTTGCAGGTCAATCTATGCTGTATTTGAATCCGACGTTAATCTGAAAGGAATCCCTGTGTATAGATTTGTTCTTCCATCCAAGGCCTTTGCCTCTCCAGTTGAAAACCCAGACAACTATTGTTTCTGCACAGAAAAAATTATCTCAAAAAATTGTACATCATATGGTGTGCTAGACATCAGCAAATGCAAAGAAGGGAGACCTGTGTACATTTCACTTCCTCATTTTCTGTATGCAAGTCCTGATGTTTCAGAACCTATTGATGGATTAAACCCAAATGAAGAAGAACATAGGACATACTTGGATATTGAACCTATAACTGGATTCACTTTACAATTTGCAAAACGGCTGCAGGTCAACCTATTGGTCAAGCCATCAGAAAAAATTCAAGTATTAAAGAATCTGAAGAGGAACTATATTGTGCCTATTCTTTGGCTTAATGAGACTGGGACCATTGGTGATGAGAAGGCAAACATGTTCAGAAGTCAAGTAACTGGAAAAATAAACCTCCTTGGCCTGATAGAAATGATCTTACTCAGTGTTGGTGTGGTGATGTTTGTTGCTTTTATGATTTCATATTGTGCATGCAGATCGAAAACAATAAAATAAGTAAGTATGTACCAAAAAATATTGCTTCAATAATATTAGCTTATATATTACTTGTTTTCACTTTATCAAAGAGAAGTTACATATTAGGCCATATATATTTCTAGACATGTCTAGCCACTGATCATTTTTAAATATAGGTAAATAAACCTATAAATATTATCACGCAGATCACTAAAGTATATCTTTAATTCTGGGAGAAATGAGATAAAAGATGTACTTGTGACCATTGTAACAATAGCACAAATAAAGCACTTGTGCCAAAGTTGTCCAAAAAACD36 Transcript Variant 3 (SEQ ID NO: 6)CTTTCAATTCCTCTGGCAACAAACCACACACTGGGATCTGACACTGTAGAGTGCTTTCTCTTCTCTTTTTTTGGGGGGGGGAGGGGGTGTGGTTGCATATTTAAACTCTCACGCATTTATGTACTGAGGACTGCAGTGTAGGACTTTCCTGCAGAATACCATTTGATCCTATTAAGAATTGTCCAAATGTTGGAGCATTTGATTGAAAAATCCTTCTTAGCCATTTTAAAGATAGCTTTCCAATGATTAGACGAATTGATTCTTTCTGTGACTCATCAGTTCATTTCCTGTAAAATTCATGTCTTGCTGTTGATTTGTGAATAAGAACCAGAGCTTGTAGAAACCACTTTAATCATATCCAGGAGTTTGCAAGAAACAGGTGCTTAACACTAATTCACCTCCTGAACAAGAAAAATGGGCTGTGACCGGAACTGTGGGCTCATCGCTGGGGCTGTCATTGGTGCTGTCCTGGCTGTGTTTGGAGGTATTCTAATGCCAGTTGGAGACCTGCTTATCCAGAAGACAATTAAAAAGCAAGTTGTCCTCGAAGAAGGTACAATTGCTTTTAAAAATTGGGTTAAAACAGGCACAGAAGTTTACAGACAGTTTTGGATCTTTGATGTGCAAAATCCACAGGAAGTGATGATGAACAGCAGCAACATTCAAGTTAAGCAAAGAGGTCCTTATACGTACAGAGTTCGTTTTCTAGCCAAGGAAAATGTAACCCAGGACGCTGAGGACAACACAGTCTCTTTCCTGCAGCCCAATGGTGCCATCTTCGAACCTTCACTATCAGTTGGAACAGAGGCTGACAACTTCACAGTTCTCAATCTGGCTGTGGCAGCTGCATCCCATATCTATCAAAATCAATTTGTTCAAATGATCCTCAATTCACTTATTAACAAGTCAAAATCTTCTATGTTCCAAGTCAGAACTTTGAGAGAACTGTTATGGGGCTATAGGGATCCATTTTTGAGTTTGGTTCCGTACCCTGTTACTACCACAGTTGGTCTGTTTTATCCTTACAACAATACTGCAGATGGAGTTTATAAAGTTTTCAATGGAAAAGATAACATAAGTAAAGTTGCCATAATCGACACATATAAAGGTAAAAGGAATCTGTCCTATTGGGAAAGTCACTGCGACATGATTAATGGTACAGATGCAGCCTCATTTCCACCTTTTGTTGAGAAAAGCCAGGTATTGCAGTTCTTTTCTTCTGATATTTGCAGGTCAATCTATGCTGTATTTGAATCCGACGTTAATCTGAAAGGAATCCCTGTGTATAGATTTGTTCTTCCATCCAAGGCCTTTGCCTCTCCAGTTGAAAACCCAGACAACTATTGTTTCTGCACAGAAAAAATTATCTCAAAAAATTGTACATCATATGGTGTGCTAGACATCAGCAAATGCAAAGAAGGGAGACCTGTGTACATTTCACTTCCTCATTTTCTCTATGCAACTCCTGATCTTTCAGAACCTATTCATGCATTAAACCCAAATCAACAACAACATACCACATACTTGGATATTGAACCTATAACTGGATTCACTTTACAATTTGCAAAACGGCTGCAGGTCAACCTATTGGTCAAGCCATCAGAAAAAATTCAAGTATTAAAGAATCTGAAGAGGAACTATATTGTGCCTATTCTTTGGCTTAATGAGACTGGGACCATTGGTGATGAGAAGGCAAACATGTTCAGAAGTCAAGTAACTGGAAAAATAAACCTCCTTGGCCTGATAGAAATGATCTTACTCAGTGTTGGTGTGGTGATGTTTGTTGCTTTTATGATTTCATATTGTGCATGCAGATCGAAAACAATAAAATAAGTAAGTATGTACCAAAAAATATTGCTTCAATAATATTAGCTTATATATTACTTGTTTTCACTTTATCAAAGAGAAGTTACATATTAGGCCATATATATTTCTAGACATGTCTAGCCACTGATCATTTTTAAATATAGGTAAATAAACCTATAAATATTATCACGCAGATCACTAAAGTATATCTTTAATTCTGGGAGAAATGAGATAAAAGATGTACTTGTGACCATTGTAACAATAGCACAAATAAAGCACTTGTGCCAAAGTTGTCCAAAAAA  CD36 Transcript Variant 4(SEQ ID NO: 7)AAGTTGCTGAGACAAGGGAAGAGAGATGAGGAACCAGAGCTTGTAGAAACCACTTTAATCATATCCAGGAGTTTGCAAGAAACAGGTGCTTAACACTAATTCACCTCCTGAACAAGAAAAATGGGCTGTGACCGGAACTGTGGGCTCATCGCTGGGGCTGTCATTGGTGCTGTCCTGGCTGTGTTTGGAGGTATTCTAATGCCAGTTGGAGACCTGCTTATCCAGAAGACAATTAAAAAGCAAGTTGTCCTCGAAGAAGGTACAATTGCTTTTAAAAATTGGGTTAAAACAGGCACAGAAGTTTACAGACAGTTTTGGATCTTTGATGTGCAAAATCCACAGGAAGTGATGATGAACAGCAGCAACATTCAAGTTAAGCAAAGAGGTCCTTATACGTACAGAGTTCGTTTTCTAGCCAAGGAAAATGTAACCCAGGACGCTGAGGACAACACAGTCTCTTTCCTGCAGCCCAATGGTGCCATCTTCGAACCTTCACTATCAGTTGGAACAGAGGCTGACAACTTCACAGTTCTCAATCTGGCTGTGGCAGCTGCATCCCATATCTATCAAAATCAATTTGTTCAAATGATCCTCAATTCACTTATTAACAAGTCAAAATCTTCTATGTTCCAAGTCAGAACTTTGAGAGAACTGTTATGGGGCTATAGGGATCCATTTTTGAGTTTGGTTCCGTACCCTGTTACTACCACAGTTGGTCTGTTTTATCCTTACAACAATACTGCAGATGGAGTTTATAAAGTTTTCAATGGAAAAGATAACATAAGTAAAGTTGCCATAATCGACACATATAAAGGTAAAAGGAATCTGTCCTATTGGGAAAGTCACTGCGACATGATTAATGGTACAGATGCAGCCTCATTTCCACCTTTTGTTGAGAAAAGCCAGGTATTGCAGTTCTTTTCTTCTGATATTTGCAGGTCAATCTATGCTGTATTTGAATCCGACGTTAATCTGAAAGGAATCCCTGTGTATAGATTTGTTCTTCCATCCAAGGCCTTTGCCTCTCCAGTTGAAAACCCAGACAACTATTGTTTCTGCACAGAAAAAATTATCTCAAAAAATTGTACATCATATGGTGTGCTAGACATCAGCAAATGCAAAGAAGGGAGACCTGTGTACATTTCACTTCCTCATTTTCTGTATGCAAGTCCTGATGTTTCAGAACCTATTGATGGATTAAACCCAAATGAAGAAGAACATAGGACATACTTGGATATTGAACCTATAACTGGATTCACTTTACAATTTGCAAAACGGCTGCAGGTCAACCTATTGGTCAAGCCATCAGAAAAAATTCAAGTATTAAAGAATCTGAAGAGGAACTATATTGTGCCTATTCTTTGGCTTAATGAGACTGGGACCATTGGTGATGAGAAGGCAAACATGTTCAGAAGTCAAGTAACTGGAAAAATAAACCTCCTTGGCCTGATAGAAATGATCTTACTCAGTGTTGGTGTGGTGATGTTTGTTGCTTTTATGATTTCATATTGTGCATGCAGATCGAAAACAATAAAATAAGTAAGTATGTACCAAAAAATATTGCTTCAATAATATTAGCTTATATATTACTTGTTTTCACTTTATCAAAGAGAAGTTACATATTAGGCCATATATATTTCTAGACATGTCTAGCCACTGATCATTTTTAAATATAGGTAAATAAACCTATAAATATTATCACGCAGATCACTAAAGTATATCTTTAATTCTGGGAGAAATGAGATAAAAGATGTACTTGTGACCATTGTAACAATAGCACAAATAAAGCACTTGTGCCAAAGTTGTCCAAAAAA  CD36 Transcript Variant 5 (SEQ ID NO: 8)ATGACATTATTAGTTCTGCCACTGGTAGGCATTAGAAGCAAGAAAAGGGAGACGGACCGAGGAAGCCACTTTGGTGAAACAAAAAGAAAAGCATTTGTTTATTTAGAACGGGCAAAATGATACGTTTCAGTGGGTGTTTTCTTTGTACTTTGATCTTTTTGTACTGATATTTAAGCTTCTGTTTTATGATCTCTTTCTAATGATAGAACCAGAGCTTGTAGAAACCACTTTAATCATATCCAGGAGTTTGCAAGAAACAGGTGCTTAACACTAATTCACCTCCTGAACAAGAAAAATGGGCTGTGACCGGAACTGTGGGCTCATCGCTGGGGCTGTCATTGGTGCTGTCCTGGCTGTGTTTGGAGGTATTCTAATGCCAGTTGGAGACCTGCTTATCCAGAAGACAATTAAAAAGCAAGTTGTCCTCGAAGAAGGTACAATTGCTTTTAAAAATTGGGTTAAAACAGGCACAGAAGTTTACAGACAGTTTTGGATCTTTGATGTGCAAAATCCACAGGAAGTGATGATGAACAGCAGCAACATTCAAGTTAAGCAAAGAGGTCCTTATACGTACAGAGTTCGTTTTCTAGCCAAGGAAAATGTAACCCAGGACGCTGAGGACAACACAGTCTCTTTCCTGCAGCCCAATGGTGCCATCTTCGAACCTTCACTATCAGTTGGAACAGAGGCTGACAACTTCACAGTTCTCAATCTGGCTGTGGCAGCTGCATCCCATATCTATCAAAATCAATTTGTTCAAATGATCCTCAATTCACTTATTAACAAGTCAAAATCTTCTATGTTCCAAGTCAGAACTTTGAGAGAACTGTTATGGGGCTATAGGGATCCATTTTTGAGTTTGGTTCCGTACCCTGTTACTACCACAGTTGGTCTGTTTTATCCTTACAACAATACTGCAGATGGAGTTTATAAAGTTTTCAATGGAAAAGATAACATAAGTAAAGTTGCCATAATCGACACATATAAAGGTAAAAGGAATCTGTCCTATTGGGAAAGTCACTGCGACATGATTAATGGTACAGATGCAGCCTCATTTCCACCTTTTGTTGAGAAAAGCCAGGTATTGCAGTTCTTTTCTTCTGATATTTGCAGGTCAATCTATGCTGTATTTGAATCCGACGTTAATCTGAAAGGAATCCCTGTGTATAGATTTGTTCTTCCATCCAAGGCCTTTGCCTCTCCAGTTGAAAACCCAGACAACTATTGTTTCTGCACAGAAAAAATTATCTCAAAAAATTGTACATCATATGGTGTGCTAGACATCAGCAAATGCAAAGAAGGGAGACCTGTGTACATTTCACTTCCTCATTTTCTGTATGCAAGTCCTGATGTTTCAGAACCTATTGATGGATTAAACCCAAATGAAGAAGAACATAGGACATACTTGGATATTGAACCTATAACTGGATTCACTTTACAATTTGCAAAACGGCTGCAGGTCAACCTATTGGTCAAGCCATCAGAAAAAATTCAAGTATTAAAGAATCTGAAGAGGAACTATATTGTGCCTATTCTTTGGCTTAATGAGACTGGGACCATTGGTGATGAGAAGGCAAACATGTTCAGAAGTCAAGTAACTGGAAAAATAAACCTCCTTGGCCTGATAGAAATGATCTTACTCAGTGTTGGTGTGGTGATGTTTGTTGCTTTTATGATTTCATATTGTGCATGCAGATCGAAAACAATAAAATAAGTAAGTATGTACCAAAAAATATTGCTTCAATAATATTAGCTTATATATTACTTGTTTTCACTTTATCAAAGAGAAGTTACATATTAGGCCATATATATTTCTAGACATGTCTAGCCACTGATCATTTTTAAATATAGGTAAATAAACCTATAAATATTATCACGCAGATCACTAAAGTATATCTTTAATTCTGGGAGAAATGAGATAAAAGATGTACTTGTGACCATTGTAACAATAGCACAAATAAAGCACTTGTGCCAAAGTTGTCCAAAAAA  CD36 Protein(SEQ ID NO: 9)MGCDRNCGLIAGAVIGAVLAVFGGILMPVGDLLIQKTIKKQVVLEEGTIAFKNWVKTGTEVYRQFWIFDVQNPQEVMMNSSNIQVKQRGPYTYRVRFLAKENVTQDAEDNTVSFLQPNGAIFEPSLSVGTEADNFTVLNLAVAAASHIYQNQFVQMTLNSLTNKSKSSMFQVRTLRELLWGYRDPFLSLVPYPVTTTVGLFYPYNNTADGVYKVFNGKDNISKVAIIDTYKGKRNLSYWESHCDMINGTDAASFPPFVEKSQVLQFFSSDICRSIYAVFESDVNLKGIPVYRFVLPSKAFASPVENPDNYCFCTEKIISKNCTSYGVLDISKCKEGRPVYISLPHFLYASPDVSEPTDGLNPNEEEHRTYLDIEPITGFTLQFAKRLQVNLLVKPSEKIQVLKNLKRNYTVPILWLNETGTIGDEKANMFRSQVTGKINLLGLIEMILLSVGVVMFVAFMISYCACRSKTIK 

Psap Peptide

Prosaposin (Psap) is the Saposin precursor protein made up ofapproximately 524-527 amino acids which includes a 16 amino acids signalpeptide. The full-length precursor polypeptide undergoesco-translational glycosylation and modification in the endoplasmicreticulum and Golgi system to yield a 70-72 kDa precursor protein. Aftertransport to the lysosome, cathepsin D participates in its proteolyticprocessing to yield intermediate molecular forms of 35 to 53 kDa andthen to a 13-kDa glycoprotein and finally to the mature 8-11 kDapartially glycosylated forms of individual Saposin molecules (O'Brien J.S., and Kishimoto Y, The FASEB J., 5: 301-8, 1991; Kishimoto Y. et al.,J. Lipid Res. 33:1255-67, 1992). Prosaposin is processed into 4 cleavageproducts: Saposins A, B, C, and D. The amino acid sequences of Psappreproprotein isoforms A, B, and C and the amino acid sequence ofcleavage product Saposin A are below:

Psap Preproprotein Isoform A (SEQ ID NO: 10)MYALFLLASLLGAALAGPVLGLKECTRGSAVWCQNVKTASDCGAVKHCLQTVWNKPTVKSLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCDWLPKPNMSASCKEIVDSYLPVILDIIKGEMSRPGEVCSALNLCESLQKHLAELNHQKQLESNKIPELDMTEVVAPFMANIPLLLYPQDGPRSKPQPKDNGDVCQDCIQMVTDIQTAVRTNSTFVQALVEHVKEECDRLGPGMADICKNYISQYSEIAIQMMMHMQPKEICALVGFCDEVKEMPMQTLVPAKVASKNVIPALELVEPIKKHEVPAKSDVYCEVCEFLVKEVTKLIDNNKTEKEILDAFDKMCSKLPKSLSEECQEVVDTYGSSILSILLEEVSPELVCSMLHLCSGTRLPALTVHVTQPKDGGFCEVCKKLVGYLDRNLEKNSTKQEILAALEKGCSFLPDPYQKQCDQFVAEYEPVLIEILVEVMDPSFVCLKIGACPSAHKPLLGTEKCIWGPSYWCQNTETAAQCNAVEHCKRHVWNPsap Preproprotein Isoform B (SEQ ID NO: 11)MYALFLLASLLGAALAGPVLGLKECTRGSAVWCQNVKTASDCGAVKHCLQTVWNKPTVKSLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCDWLPKPNMSASCKEIVDSYLPVILDIIKGEMSRPGEVCSALNLCESLQKHLAELNHQKQLESNKIPELDMTEVVAPFMANIPLLLYPQDGPRSKPQPKDNGDVCQDCIQMVTDIQTAVRTNSTFVQALVEHVKEECDRLGPGMADICKNYISQYSEIAIQMMMHMQDQQPKEICALVGFCDEVKEMPMQTLVPAKVASKNVIPALELVEPIKKHEVPAKSDVYCEVCEFLVKEVTKLIDNNKTEKEILDAFDKMCSKLPKSLSEECQEVVDTYGSSILSILLEEVSPELVCSMLHLCSGTRLPALTVHVTQPKDGGFCEVCKKLVGYLDRNLEKNSTKQEILAALEKGCSFLPDPYQKQCDQFVAEYEPVLIEILVEVMDPSFVCLKIGACPSAHKPLLGTEKCIWGPSYWCQNTETAAQCNAVEHCKRHVWNPsap Preproprotein Isoform C (SEQ ID NO: 12)MYALFLLASLLGAALAGPVLGLKECTRGSAVWCQNVKTASDCGAVKHCLQTVWNKPTVKSLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCDWLPKPNMSASCKEIVDSYLPVILDIIKGEMSRPGEVCSALNLCESLQKHLAELNHQKQLESNKIPELDMTEVVAPFMANIPLLLYPQDGPRSKPQPKDNGDVCQDCIQMVTDIQTAVRTNSTFVQALVEHVKEECDRLGPGMADICKNYISQYSEIAIQMMMHMDQQPKEICALVGFCDEVKEMPMQTLVPAKVASKNVIPALELVEPIKKHEVPAKSDVYCEVCEFLVKEVTKLIDNNKTEKEILDAFDKMCSKLPKSLSEECQEVVDTYGSSILSILLEEVSPELVCSMLHLCSGTRLPALTVHVTQPKDGGFCEVCKKLVGYLDRNLEKNSTKQEILAALEKGCSFLPDPYQKQCDQFVAEYEPVLIEILVEVMDPSFVCLKIGACPSAHKPLLGTEKCIWGPSYWCQNTETAAQCNAVEHCKRHVWN Saposin A (SEQ ID NO: 13)SLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCDWLPKPNMSASCKEIVDSYLPVILDIIKGEMSRPGEVCSALNLCES

Aspects of the disclosure relate to a Psap peptide and uses thereof.Psap peptides comprise sequences that were originally derived fromfragments of Saposin A. It was shown previously that fragments ofSaposin A consisting of as few as 4 amino acids, and variants of thesefragments, had anti-angiogenic and anti-cancer activity. Psap peptidesand methods of making Psap peptides are known in the art (see, e.g., PCTpublications WO2009002931 and WO/2011/084685; PCT applicationPCT/US2012/71424, published as PCT publication WO/2013/096868, and U.S.patent application Ser. Nos. 12/640,788 and 13/516,511, all of which areincorporated herein by reference in their entirety).

In some embodiments, a Psap peptide comprises the amino acid sequenceCDWLPK (SEQ ID NO: 1), DWLPK (SEQ ID NO: 2), or DWLP (SEQ ID NO: 3), oran amino acid substitution variant thereof, wherein the amino acidsubstitution is:

a) Tyrosine (Y) for Tryptophan (W);

b) an amino acid substitution for Leucine (L) selected from Valine (V),Alanine (A) or Glycine (G), or a non-canonical amino acid of similarsize, or a derivative thereof;

c) Arginine (R) for Lysine (K);

d) a D-isomer of Aspartic Acid (D) for an L-isomer of Aspartic Acid (D)and/or a D-isomer of Leucine (L) for a L-isomer of Leucine (L);

e) a D-isomer of Tryptophan (W) for an L-isomer of Tryptophan (W) and/ora D-isomer of Proline (P) for an L-isomer of Proline (P); orcombinations thereof. In some embodiments, a Psap peptide comprises theamino acid sequence CDWLPK (SEQ ID NO: 1), DWLPK (SEQ ID NO: 2), or DWLP(SEQ ID NO: 3).

It is to be understood that a Psap peptide can be of any length. In someembodiments, the Psap peptide is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95 or 100 or more amino acids in length. In some embodiments, thePsap peptide is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 33, 34, 35, 36,37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200,300, 400, 500 or fewer amino acids in length. In some embodiments, thePsap peptide is 4-500, 4-400, 4-300, 4-200, 4-100, 4-90, 4-80, 4-70,4-60, 4-50, 4-40, 4-30, 4-25, 4-20, 5-500, 5-400, 5-300, 5-200, 5-100,5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, 5-25, 5-20, 6-500, 6-400,6-300, 6-200, 6-100, 6-90, 6-80, 6-70, 6-60, 6-50, 6-40, 6-30, 6-25, or6-20 amino acids in length.

It is to be understood that amino acids flanking the CDWLPK (SEQ ID NO:1), DWLPK (SEQ ID NO: 2), or DWLP (SEQ ID NO: 3) may be the naturallyflanking amino acids present in Saposin A or Prosaposin (e.g.,LEKTCDWLPKPNMS (SEQ ID NO: 14), the underlined amino acids are the aminoacids naturally flanking the DWLP (SEQ ID NO: 3) sequence in Saposin A).Accordingly, in some embodiments, the Psap peptide comprises the aminoacid sequence DWLPKPNMS (SEQ ID NO: 15), CDWLPKPNM (SEQ ID NO: 16),TCDWLPKPN (SEQ ID NO: 17), KTCDWLPKP (SEQ ID NO: 18), EKTCDWLPK (SEQ IDNO: 19), LEKTCDWLP (SEQ ID NO: 20) or an amino acid substitution variantthereof wherein the substitution occurs in CDWLPK (SEQ ID NO: 1), DWLPK(SEQ ID NO: 2), or DWLP (SEQ ID NO: 3). Other examples of Psap peptidesinclude without limitation, DWLPKPNMS (SEQ ID NO: 21), CDWLPKPNM (SEQ IDNO: 22), TCDWLPKPN (SEQ ID NO: 23), KTCDWLPKP (SEQ ID NO: 24), EKTCDWLPK(SEQ ID NO: 25), and LEKTCDWLP (SEQ ID NO: 26). Other Psap peptideexamples include, without limitation, DWLPKPNM (SEQ ID NO: 27), CDWLPKPN(SEQ ID NO: 28), TCDWLPKP (SEQ ID NO: 29), KTCDWLPK (SEQ ID NO: 30),EKTCDWLP (SEQ ID NO: 31), DWLPKPN (SEQ ID NO: 32), CDWLPKP (SEQ ID NO:33), TCDWLPK (SEQ ID NO: 34), KTCDWLP (SEQ ID NO: 35), DWLPKP (SEQ IDNO: 36), CDWLPK (SEQ ID NO: 1), TCDWLP (SEQ ID NO: 37), DWLPK (SEQ IDNO: 2), CDWLP (SEQ ID NO: 38), and DWLP (SEQ ID NO: 3).

It is also to be understood that amino acids flanking CDWLPK (SEQ ID NO:1), DWLPK (SEQ ID NO: 2), or DWLP (SEQ ID NO: 3) need not be thenaturally flanking amino acids present in Saposin A or Prosaposin, butcan instead be any amino acid. Thus, a Psap peptide can include anynumber and identity of flanking amino acids. In some embodiments, theflanking amino acids may comprise an antibody or antibody Fc domain,serum transferrin or portions thereof, albumin, or transthyretin (see,e.g., G. M. Subramanian, (2007), Nature Biotechnology 25, 1411-141).

Psap peptides can be synthesized using any method known in the art.Exemplary methods of synthesis include, but are not limited to,recombinant synthesis, liquid-phase synthesis, Solid-phase synthesis,chemical ligation (see, e.g., Molecular Cloning: A Laboratory Manual, J.Sambrook, et al., eds., Third Edition, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 2001; Current Protocols in MolecularBiology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York;Schnolzer, M. A., P.; Jones, A.; Alewood, D.; Kent, S. B. H. (2007). “InSitu Neutralization in Boc-chemistry Solid Phase Peptide Synthesis”.Int. J. Peptide Res. Therap. 13 (1-2): 31-44; Albericio, F. (2000).Solid-Phase Synthesis: A Practical Guide (1 ed.). Boca Raton: CRC Press.p. 848; and Nilsson B L, Soellner M B, Raines R T (2005). “ChemicalSynthesis of Proteins”. Annu. Rev. Biophys. Biomol. Struct. 34: 91-118;and U.S. Pat. Nos. 4,749,742, 4,794,150, 5,552,471, 5,637,719,6,001,966, 7,038,103, 7,094,943, 7,176,282, and 7,645,858, the entiretyof which are incorporated herein by reference).

In some embodiments, the Psap peptide may be modified, for example,through oligomerization or polymerization (e.g., dimers, timer,multimers, etc.), modifications of amino acid residues or peptidebackbone, cross-linking, cyclization, conjugation, pegylation,glycosylation, acetylation, phosphorylation, fusion to additionalheterologous amino acid sequences (for example, an antibody or antibodyFc domain, serum transferrin or portions thereof, albumin, ortransthyretin), or other modifications that substantially alter thestability, solubility, or other properties of the peptide whilesubstantially retaining or enhancing therapeutic activity. Conjugationmay be, e.g., to a polymer. Suitable polymers include, for example,polyethylene glycol (PEG), polyvinyl pyrrolidone, polyvinyl alcohol,polyamino acids, divinylether maleic anhydride,N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran derivativesincluding dextran sulfate, polypropylene glycol, polyoxyethylatedpolyol, heparin, heparin fragments, polysaccharides, cellulose andcellulose derivatives, including methylcellulose and carboxymethylcellulose, starch and starch derivatives, polyalkylene glycol andderivatives thereof, copolymers of polyalkylene glycols and derivativesthereof, polyvinyl ethyl ethers, andα,β-Poly[(2-hydroxyethyl)-DL-aspartamide, and the like, or mixturesthereof. Conjugation may be through a linker, e.g., a peptide orchemical linker. Methods of modifying peptides are well known in the art(see, e.g., U.S. Pat. Nos. 5,180,816, 5,596,078, 5,990,273, 5,766,897,5,856,456, 6,423,685, 6,884,780, 7,610,156, 7,256,258, 7,589,170 and7,022,673, and PCT publication WO 2010/014616, the contents of which areincorporated herein by reference).

In some embodiments, the Psap peptide is a cyclic peptide. Cyclicpeptides are polypeptide chains whose amino and carboxyl termini arelinked together with a peptide bond or other covalent bond, forming acircular chain. In one embodiment, the peptide contains amino andcarboxyl terminal cysteine amino acid residues. Cysteines facilitate S—Sdisulfide bond formation. In one embodiment, the peptide containsadditional cysteine amino acid residues, wherein the cysteine amino acidresidues are near the termini but not necessarily at the very end. Insome embodiments, the cysteine amino acid residues are within the fiveamino acid residues of the termini of the peptide. Methods of design andsynthesis of cyclic peptides are well known in the art, e.g. asdescribed in U.S. Pat. Nos. 5,596,078; 5,990,273; 7,589,170 and U.S.Patent Application No. 20080287649.

In some embodiments, the Psap peptide is functionally modified toenhance stability. In some embodiments, the Psap peptide comprises anN-terminal acetyl group and/or a C terminal amide group. In someembodiments, the Psap peptide comprises an N-terminal acetyl group and aC terminal amide group. In some embodiments, the Psap peptide isAc-dW1P-Amide or Ac-DWLP-Amide (Ac=acetyl group, lower case D and Lindicate D-amino acids, SEQ ID NOs: 39 and 40, respectively). In someembodiments, chemical modifications to the Psap peptide include, but arenot limited to the inclusion of, alkyl, alkoxy, hydroxyalkyl,alkoxyalkyl, alkoxycarbonyl, alkenyl, alkynyl, cycloalkyl, amino,alkylamino, aminoalkyl, dialkylamino, aminodialkyl, halogen, heteroatom,carbocycle, carbocyclyl, carbocyclo, carbocyclic, aryl, aralkyl,aralkoxy, aryloxyalkyl, heterocycle, heterocyclyl, heterocyclic,heteroaryl, and/or aliphatic groups.

Psap peptides also encompass peptidomimetics (e.g., D-peptides, βpeptides and peptoids). The peptidomimetics utilized can encompass theentire length of the Psap peptide, or only a portion of the Psappeptide. Peptidomimetics may include, e.g., D-amino acids, reduced amidebonds for the peptide backbone, and non-peptide bonds to link the sidechains, pyrrolinone and sugar mimetics. The design and synthesis ofsugar scaffold peptide mimetics are described by Hirschmann et al. (J.Med. Chem., 1996, 36, 2441-2448, which is incorporated herein byreference in its entirety). Further, pyrrolinone-based peptidomimeticsare also described (see, for example, Smith et al., J. Am. Chem. Soc.2000, 122, 11037-11038, which is incorporated herein by reference in itsentirety). In some embodiments the Psap peptide is in the form of apeptoid (U.S. Pat. No. 5,811,387; Simon et al. Proceedings of theNational Academy of Sciences USA, (1992), 89(20), 9367-9371). In someembodiments, peptoids are poly-N-substituted glycines. In peptoids theside chain is connected to the nitrogen of the peptide backbone, insteadof the α-carbon as in peptides. In some embodiments the peptoid containsnitroaromatic monomer units (Fowler et al., J Org Chem. 2009 Feb. 20;74(4):1440-9). In some embodiments, the peptoid is N-substituted withalpha-chiral aromatic side chains (Gorske et al., J Am Chem Soc. 2006Nov. 8; 128(44):14378-87) at one or more residues. In some embodiments,the Psap peptide comprises a peptoid region (i.e., containing one ormore side chains connected to the nitrogen of the peptide backbone) anda peptide region (i.e., containing one or more side chains connected tothe α-carbon).

Psap Amino Acid Substitutions

In some embodiments, a Psap peptide comprises an amino acid substitutionvariant of CDWLPK (SEQ ID NO: 1), DWLPK (SEQ ID NO: 2), or DWLP (SEQ IDNO: 3), wherein the amino acid substitution is:

a) Tyrosine (Y) for Tryptophan (W);

b) an amino acid substitution for Leucine (L) selected from Valine (V),Alanine (A) or Glycine (G), or a non-canonical amino acid of similarsize, or a derivative thereof;

c) Arginine (R) for Lysine (K);

d) a D-isomer of Aspartic Acid (D) for an L-isomer of Aspartic Acid (D)and/or a D-isomer of Leucine (L) for a L-isomer of Leucine (L);

e) a D-isomer of Tryptophan (W) for an L-isomer of Tryptophan (W) and/ora D-isomer of Proline (P) for an L-isomer of Proline (P); orcombinations thereof.

Conservative amino acid substitutions can be replacement of one aminoacid residue with an amino acid residue having a side chain with asimilar charge, size, polarity, hydrophobicity, or combination thereof.Families of amino acid residues having side chains with similar chargeshave been defined in the art. These families include amino acids withbasic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

Conservative amino acid substitutions typically do not change theoverall structure of the peptide and/or the type of amino acid sidechains available for forming van der Waals bonds with a binding partner.In some embodiments, a conservative substitution for Leucine is Valine.In some embodiments, a conservative substitution for Leucine is Valineor Alanine.

In some embodiments, conservative or non-conservative substitutions forLeucine are contemplated. In some embodiments, the substitution forLeucine is Valine, Glycine or Alanine. In some embodiments, asubstitution for Leucine is Glycine. In some embodiments, a substitutionfor Leucine is Glycine or Valine. In some embodiments, the amino acidsubstitution is a Tyrosine (Y) for a Tryptophan (W).

Exemplary amino acid substitution variants include, but are not limitedto, DWAP (SEQ ID NO: 41), DYLPK (SEQ ID NO: 42), DWVPK (SEQ ID NO: 43),DWLPR(SEQ ID NO: 44), DWAPK (SEQ ID NO: 45), and DYLP (SEQ ID NO: 46).

Substitution with a non-canonical amino acid is also contemplatedherein. In some embodiments, Leucine is substituted with a non-canonicalamino acid. In some embodiments, the non-canonical amino acid substitutefor Leucine has a similar size to Leucine, Valine, Alanine, or Glycine.Examples of non-canonical amino acids include azidoalanine,azidohomoalanine, azidonorvaline, azidonorleucine, azidonorvaline,homoallyglycine, homoproparglycine, norvaline, norleucine,cis-crotyiglycine, trans-crotylglycine, 2-aminoheptanoic acid,2-butynyiglycine, allyglycine, 3-(1-naphthyl)alanine, 3-(2-naphthyl)alanine, p-ethynyl-phenylalanine, p-propargly-oxy-phenylalanine,m-ethynyl-phenylalanine, 3-(6-chloroindolyl)alanine,3-(6-bromoindolyl)alanine, 3-(5-bromoindolyl)alanine, azidohomoalanine,homopropargylglycine, p-chlorophenylalanine, α-aminocaprylic acid,methylvaline, methylleucine, or sarcosine. In some embodiments, Leucineis substituted with a non-canonical amino acid selected frommethylvaline, methylleucine, or sarcosine. Non-canonical amino acids andmethods of synthesis thereof are well known in the art (see, e.g., U.S.Patent Publications 2010-0247433, 2008-0214439, 2004-0053390, and2004-0058415; PCT publication WO 03/073238; and U.S. Pat. No. 6,586,207,all of which are incorporated herein by reference).

Amino acid substitution can be achieved during chemical synthesis of thepeptide by adding the desired substitute amino acid at the appropriatesequence in the synthesis process. Alternatively, molecular biologymethods can be used. Non-conservative substitutions are also encompassedto the extent that they substantially retain the activities of thosepeptides described herein.

As previously described, Psap peptides comprising CDWLPK (SEQ ID NO: 1),DWLPK (SEQ ID NO: 2), or DWLP (SEQ ID NO: 3) having D-amino acidsubstitutions were also shown to have a desired therapeutic activity(see PCT application PCT/US2012/71424, published as PCT publicationWO/2013/096868). As such, amino acid substitution variants resultingfrom substitution of one or more D-amino acids for the like L-amino acidare contemplated herein. In some embodiments, one D-amino acidsubstitution is present. In some embodiments, 2 or more D-amino acidsubstitutions are present. In some embodiments, 3, 4, or 5 D-amino acidsubstitutions are present. In some embodiments, the D-amino acidsubstitutions are evenly spaced, e.g., every other amino acid, of the4-6 mer. In some embodiments, the D-amino acid substitution is forTryptophan (W) and/or Proline (P). In some embodiments, the D-amino acidsubstitution is for Aspartic Acid (D) and/or Leucine (L)). The L and Dconvention for amino acid configuration refers not to the opticalactivity of the amino acid itself, but rather to the optical activity ofthe isomer of glyceraldehyde from which that amino acid can, in theory,be synthesized (D-glyceraldehyde is dextrorotary; L-glyceraldehyde islevorotary). Exemplary D amino acid substitutions include dW1P and DwLp(lower case D and L indicate D-amino acids, SEQ ID NOs: 47 and 48,respectively).

Assay

Aspects of the disclosure relate to performing an assay to determine alevel of CD36 in a sample. Any assay known in the art can be used formeasuring a CD36 level (see, e.g., Molecular Cloning: A LaboratoryManual, J. Sambrook, et al., eds., Third Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 2001, Current Protocols inMolecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc.,New York. Microarray technology is described in Microarray Methods andProtocols, R. Matson, CRC Press, 2009, or Current Protocols in MolecularBiology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., NewYork). The level of CD36 can be an mRNA level and/or a protein level. Insome embodiments, the level of CD36 is a protein level. Assays fordetecting CD36 mRNA include, but are not limited to, Northern blotanalysis, RT-PCR, sequencing technology, RNA in situ hybridization(using e.g., DNA or RNA probes to hybridize to RNA molecules present inthe sample), in situ RT-PCR (e.g., as described in Nuovo G J, et al. AmJ Surg Pathol. 1993, 17: 683-90; Komminoth P, et al. Pathol Res Pract.1994, 190: 1017-25), and oligonucleotide microarray (e.g., byhybridization of polynucleotide sequences derived from a sample tooligonucleotides attached to a solid surface (e.g., a glass wafer) withaddressable locations, such as an Affymetrix microarray (Affymetrix®,Santa Clara, Calif.)). Methods for designing nucleic acid bindingpartners, such as probes, are well known in the art. In someembodiments, the nucleic acid binding partners bind to a part of or anentire nucleic acid sequence of CD36, the sequence being identifiablewith the CD36 sequences provided herein.

Assays for detecting CD36 protein levels include, but are not limitedto, immunoassays (also referred to herein as immune-based orimmuno-based assays, e.g., Western blot, immunohistochemistry and ELISAassays), Mass spectrometry, and multiplex bead-based assays. Such assaysfor protein level detection are well-known in the art. Binding partnersfor protein detection can be designed using methods known in the art andas described herein. In some embodiments, the CD36 protein bindingpartners, e.g., anti-CD36 antibodies, bind to a part of or an entireamino acid sequence of the CD36 protein. Other examples of proteindetection and quantitation methods include multiplexed immunoassays asdescribed for example in U.S. Pat. Nos. 6,939,720 and 8,148,171, andpublished US Patent Application No. 2008/0255766, and proteinmicroarrays as described for example in published US Patent ApplicationNo. 2009/0088329.

In some embodiments, the sample obtained from a subject is a tumorbiopsy and the assay for detecting CD36 protein levels is animmuno-based assay performed on the tumor biopsy.

Any suitable binding partner for CD36 is contemplated for detection of aCD36 level. In some embodiments, the binding partner is any moleculethat binds specifically to a CD36 protein. As described herein, “bindsspecifically to a CD36 protein” means that the molecule is more likelyto bind to a portion of or the entirety of a CD36 protein than to aportion of or the entirety of a non-CD36 protein. In some embodiments,the binding partner is an antibody or antigen-binding fragment thereof,such as Fab, F(ab)2, Fv, single chain antibodies, Fab and sFabfragments, F(ab′)2, Fd fragments, scFv, or dAb fragments. Methods forproducing antibodies and antigen-binding fragments thereof are wellknown in the art (see, e.g., Sambrook et al, “Molecular Cloning: ALaboratory Manual” (2nd Ed.), Cold Spring Harbor Laboratory Press(1989); Lewin, “Genes IV”, Oxford University Press, New York, (1990),and Roitt et al., “Immunology” (2nd Ed.), Gower Medical Publishing,London, New York (1989), WO2006/040153, WO2006/122786, andWO2003/002609). Binding partners also include other peptide moleculesand aptamers that bind specifically to CD36. Methods for producingpeptide molecules and aptamers are well known in the art (see, e.g.,published US Patent Application No. 2009/0075834, U.S. Pat. Nos.7,435,542, 7,807,351, and 7,239,742).

Commercially available CD36 antibodies include, for example, N-15, SMφ,L-17, ME542, H300, 185-1G2, and V-19 from Santa Cruz Biotechnology(Catalog numbers sc-5522, sc-7309, sc-13572, sc-5523, sc-9154, sc-21772,and sc-7641, respectively), JC63.1, FA6-152, and anti-CD36 from Abcam(Catalog numbers ab23680, ab17044, and ab78054, respectively).

In some embodiments, the binding partner is any molecule that bindsspecifically to a CD36 mRNA. As described herein, “binds specifically toa CD36 mRNA” means that the molecule is more likely to bind to a portionof or the entirety of the CD36 mRNA (e.g., by complementarybase-pairing) than to a portion of or the entirety of a non-CD36 mRNA orother non-CD36 nucleic acid. In some embodiments, the binding partnerthat binds specifically to a CD36 mRNA is a nucleic acid, e.g., a probe.Binding partners can be designed using the nucleotide and amino acidsequences of CD36, which are provided herein. In some embodiments, aCD36 binding partner may comprise a detectable label, such as anenzymatically active group, a fluorescent molecule, a chromophore, aluminescent molecule, a specifically bindable ligand, or a radioisotope.In some embodiments, a second binding partner specific for the CD36binding partner is also contemplated, such as a secondary antibody.

Sample

Aspects of the disclosure relate to determining a level of CD6 in asample obtained from a subject. In some embodiments, the sample obtainedfrom a subject is a tumor sample. As used herein, a tumor sample maycomprise, e.g., a tumor cell, a population of tumor cells, a fragment ofa tumor (e.g., a biopsy), or an entire tumor. In some embodiments, thetumor sample is a tumor biopsy. In some embodiments, the tumor samplecomprises circulating tumor cells. In some embodiments, the tumor samplecomprises ascites. In some embodiments, the tumor sample comprisespleural fluid. The tumor sample may contain non-tumor cells or non-tumortissue (e.g., a biopsy that contains normal tissue surrounding a tumorfragment). In some embodiments, the sample may be a tissue or fluidsample obtained from a subject. Examples of fluid samples are blood,plasma, serum, and urine.

Subjects

Aspects of the disclosure relate to subjects, such as human subjects,with cancer. Any type of cancer is contemplated herein, including, butnot limited to, leukemias, lymphomas, myelomas, carcinomas, metastaticcarcinomas, sarcomas, adenomas, nervous system cancers and genitourinarycancers. Exemplary cancer types include adult and pediatric acutelymphoblastic leukemia, acute myeloid leukemia, adrenocorticalcarcinoma, AIDS-related cancers, anal cancer, cancer of the appendix,astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer,bone cancer, osteosarcoma, fibrous histiocytoma, brain cancer, brainstem glioma, cerebellar astrocytoma, malignant glioma, ependymoma,medulloblastoma, supratentorial primitive neuroectodermal tumors,hypothalamic glioma, breast cancer, male breast cancer, bronchialadenomas, Burkitt lymphoma, carcinoid tumor, carcinoma of unknownorigin, central nervous system lymphoma, cerebellar astrocytoma,malignant glioma, cervical cancer, childhood cancers, chroniclymphocytic leukemia, chronic myelogenous leukemia, chronicmyeloproliferative disorders, colorectal cancer, cutaneous T-celllymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewingfamily tumors, extracranial germ cell tumor, extragonadal germ celltumor, extrahepatic bile duct cancer, intraocular melanoma,retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinalstromal tumor, extracranial germ cell tumor, extragonadal germ celltumor, ovarian germ cell tumor, gestational trophoblastic tumor, glioma,hairy cell leukemia, head and neck cancer, hepatocellular cancer,Hodgkin lymphoma, non-Hodgkin lymphoma, hypopharyngeal cancer,hypothalamic and visual pathway glioma, intraocular melanoma, islet celltumors, Kaposi sarcoma, kidney cancer, renal cell cancer, laryngealcancer, lip and oral cavity cancer, small cell lung cancer, non-smallcell lung cancer, primary central nervous system lymphoma, Waldenstrommacroglobulinema, malignant fibrous histiocytoma, medulloblastoma,melanoma, Merkel cell carcinoma, malignant mesothelioma, squamous neckcancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosisfungoides, myelodysplastic syndromes, myeloproliferative disorders,chronic myeloproliferative disorders, nasal cavity and paranasal sinuscancer, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer,ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer,pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorialprimitive neuroectodermal tumors, pituitary cancer, plasma cellneoplasms, pleuropulmonary blastoma, prostate cancer, rectal cancer,rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma, uterinesarcoma, Sezary syndrome, non-melanoma skin cancer, small intestinecancer, squamous cell carcinoma, squamous neck cancer, supratentorialprimitive neuroectodermal tumors, testicular cancer, throat cancer,thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer,trophoblastic tumors, urethral cancer, uterine cancer, uterine sarcoma,vaginal cancer, vulvar cancer, or Wilms tumor.

In some embodiments, the cancer is prostate cancer, breast cancer,ovarian cancer, lung cancer, leukemia, pancreatic cancer, glioblastomamultiforme, astrocytoma or melanoma. In some embodiments, the cancer isprostate cancer, breast cancer, lung cancer, leukemia, pancreaticcancer, glioblastoma multiforme, astrocytoma or melanoma. In someembodiments, the cancer is pancreatic cancer, ovarian cancer, breastcancer, prostate cancer, melanoma cancer, or lung cancer.

Control and Control Level

Aspects of the disclosure relate to comparison of a CD36 level in asample with a control level. In some embodiments, the control level is alevel of CD36 in a cell, tissue or fluid obtained from a healthy subjector population of healthy subjects. As used herein, a healthy subject isa subject that is apparently free of disease and has no history ofdisease, such as cancer.

In some embodiments, the control level is determined from a sampleobtained from a subject having cancer. Accordingly, in some embodimentsthe control level is obtained from the same subject from whom the sampleis obtained. In some embodiments, a control level is a level of CD36from a non-cancerous cell or tissue obtained from the subject having thecancer.

In some embodiments, a control level is a level of CD36 that isundetectable or below a background/noise level obtained using standardmethods of detection (e.g., Western blot or immunohistochemistry).

The disclosure also involves comparing the level of CD36 in a samplefrom the subject with a predetermined level or value, such that acontrol level need not be measured every time. The predetermined levelor value can take a variety of forms. It can be single cut-off value,such as a median or mean. It can be established based upon comparativegroups, such as where one defined group is known not to respond totreatment with a Psap peptide and another defined group is known to beresponsive to treatment with a Psap peptide. It can be a range, forexample, where the tested population is divided equally (or unequally)into groups, such as a unresponsive to treatment with a Psap peptide,somewhat responsive to treatment with a Psap peptide, and highlyresponsive to treatment with a Psap peptide, or into quadrants, thelowest quadrant being subjects with no response to treatment with a Psappeptide and the highest quadrant being subjects with the highestresponse to treatment with a Psap peptide response.

The predetermined value can depend upon the particular populationselected. For example, an apparently healthy (no detectable cancer andno prior history of cancer) will have a different ‘normal’ range of CD36than will a population the members of which have cancer but are knownnot to respond to treatment with a Psap peptide. Accordingly, thepredetermined values selected may take into account the category inwhich a subject falls. Appropriate ranges and categories can be selectedwith no more than routine experimentation by those of ordinary skill inthe art.

EXAMPLES Example 1 Methods Cell Lines and Primary Cells

The cell line PC3 was previously described (Kang et al. PNAS. 2009;106:12115-20). PC3 cells were cultured in RPMI with 10% FBS. Humanbreast cancer cell lines MDA-MB-231 and MCF-7 were described previously(Ryu et al. PLoS one, 6, 2011). The murine Lewis lung carcinoma cellline LLCs (provided by Lea Eisenbach, Wiesmann Institute of Science,Rehovot, Israel) stably expressing RFP and firefly luciferase (Gupta GP, Massague J. Cancer metastasis: building a framework. Cell. 2006;127:679-95; Gao D, Nolan D J, Mellick A S, Bambino K, McDonnell K,Mittal V. Endothelial progenitor cells control the angiogenic switch inmouse lung metastasis. Science. 2008; 319:195-8; and Joyce J A, PollardJ W. Microenvironmental regulation of metastasis. Nat Rev Cancer. 2009;9:239-52), was cultured in DMEM supplemented with 10% fetal bovineserum. B16 melanoma cells, LNCaP prostate cancer cells, AsPc1 pancreaticcancer cells, and ID8 ovarian cancer cells were previously described(Overwijk W W et al. B16 as a mouse model for human melanoma. CurrProtoc Immunol. 2001, May; Chapter 20:Unit 20.1; Horoszewicz J S, LeongS S, Kawinski E et al. LNCaP model of human prostatic carcinoma. CancerRes. 1983, April; 43(4):1809-18.; Chen W H, et al. Human pancreaticadenocarcinoma: in vitro and in vivo morphology of a new tumor lineestablished from ascites. In Vitro 18: 24-34, 1982; and Roby K F, et al.Development of a syngeneic mouse model for events related to ovariancancer. Carcinogenesis. 2000, 21:585-591). Primary ovarian cancer cellswere derived from ovarian cancer patient ascites.

Western Blot Analysis

Cells were homogenized in lysis buffer (BioRad) containing proteaseinhibitors (Roche Applied Science). Samples were boiled in 1×SDSsampling buffer, and loaded onto 4-20% gradient Bis-Tris NuPAGE gels(Invitrogen). Western blotting was performed using antibodies specificfor CD36 (AbCam, ab78054) or β-actin (Sigma-Aldrich).

In Vitro Cell Proliferation Assays

Cell proliferation was measured using the MTT(3-{4,5-Dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide,Sigma-Aldrich) assay. Cells were seeded in 50 uL growth medium in 96well culture plates and allowed to attach overnight. 50 uL of growthmedium plus two-fold concentrated treatment reagents were then added.After each treatment time point, 10 uL of 5% MTT solution (buffered inPBS) was added to each well. Plates were incubated for an additional 4 hat 37° C. to allow MTT to be metabolically converted into formazancrystals at cell mitochondria. The formazan crystals were finallysolubilized by adding 100 ul of 10% Sodium Dodecyl Sulphate in 50%N—N-Dimethylformamide to each microplate well. Absorbances at 550 and680 nm (corresponding to formazan salt and reference wavelengths,respectively) were measured using a colorimetry microplate reader. Wellscontaining only complete medium were used as controls. Each experimentwas performed twice, using six replicates for each drug concentration.

Results

It was hypothesized that Tsp-1 upregulated by a Psap peptide may beacting directly on the cancer cells, rather than only through anindirect anti-angiogenic mechanism. To test this, LLC cells were treatedwith either recombinant Tsp-1 or DWLPK (SEQ ID NO: 2) Psap peptide andcell proliferation was measured using an MTT assay. It was found thatTsp-1 was capable of decreasing cell proliferation, while the Psappeptide did not affect cell proliferation (FIG. 1A). This supports thehypothesis that Tsp-1 can act directly on cancer cells, as this assaywas performed in vitro in the absence of any blood vessels. Theseresults also show that the Psap peptide alone does not appear affectcancer cell proliferation, supporting the hypothesis that Psap peptidesmay indirectly treat cancer through upregulation of Tsp-1. LLC cellswere shown to express CD36, a receptor for Tsp-1, indicating that Tsp-1may act directly on cancer cells through CD36 (FIG. 1B).

CD36 levels were measured in other cell lines to see if CD36 was alsoexpressed by other cancer types. CD36 levels were measured in breastcancer (MDA-231, MCF-7), ovarian cancer (ID8), melanoma (B16), prostatecancer (PC3 and LNCaP), and lung cancer (LLC) cell lines by western blotanalysis. It was found that CD36 protein was detectable in all celllines tested, with particularly high levels of CD36 detectable inMDA-231, MCF-7, PC3, and LLC cell lines (FIG. 2). MDA-231, ID8, B16,PC3, and LLC cells have been shown previously to respond to Treatmentwith a Psap peptide in vivo.

The pancreatic cell line AsPc1 was also examined and found to expressCD36.

CD36 levels were also measured in primary ovarian cancer cells derivedfrom patients with ascites. CD36 protein was detectable in all primaryovarian cancer cells tested (FIG. 3).

Example 2 Methods Mice and Cell Lines

All animal work is conducted in accordance with a protocol approved bythe Institutional Animal Care and Use Committee. Wild type C57BL/6J, andGFP transgenic C57BL/6-Tg (ACTB-EGFP) iOsb/J are obtained from TheJackson Laboratory (Bar Harbor, Me.). CB-17 SCID mice are obtained fromCharles River (Wilmington, Mass.).

The cell lines PC3 and PC3M-LN4 are previously described (14). Humanbreast cancer cell lines MDA-MB-231 and MDA-MB-LM2 are describedpreviously (Ryu et al. PLoS one, 6, 2011). The murine Lewis lungcarcinoma cell line LLCs/D122 (provided by Lea Eisenbach, WiesmannInstitute of Science, Rehovot, Israel) stably expressing RFP and fireflyluciferase (Gupta G P, Massague J. Cancer metastasis: building aframework. Cell. 2006; 127:679-95; Gao D, Nolan D J, Mellick A S,Bambino K, McDonnell K, Mittal V. Endothelial progenitor cells controlthe angiogenic switch in mouse lung metastasis. Science. 2008;319:195-8; and Joyce J A, Pollard J W. Microenvironmental regulation ofmetastasis. Nat Rev Cancer. 2009; 9:239-52), are cultured in DMEMsupplemented with 10% fetal bovine serum.

Tissue Microarrays and Immunohistochemistry

Archival specimens (radical prostatectomy specimens, or biopsies ofmetastases) are retrieved from files of Department of Pathology, TheGade Institute, Haukeland University Hospital. Formalin fixedprostatectomy specimens were paraffin embedded and studied by wholemount step sections at 5 mm intervals. Tissue microarrays (TMAs) wereconstructed selecting three tissue cores (0.6 mm in diameter) from thearea of highest tumor grade in each case.

Thin paraffin sections (5 um) from the TMA paraffin block are dewaxedwith xylene/ethanol before heat induced microwave epitope retrieval incitrate buffer (pH 6.0) for 20 minutes, and incubated with a CD36antibody for 60 minutes at room temperature. Immunostaining is performedon the DAKO Autostainer with the EnVision chain polymer method (DakoCytomation, Copenhagen, Denmark) as detection system. Antigenlocalization is achieved using the DAB diaminobenzidine peroxidasereaction, counterstained with hematoxylin.

Immunostaining is estimated semiquantitatively, and a staining index(SI) obtained as a product of staining intensity (0-3) and proportion ofimmunopositive tumor cells (<10%=1, 10-50%=2, >50%=3), is calculated.The staining index (range 0-9) is a categorical scale, where somevariation within each category is expected.

Knockdown of CD36 in Tumor Cells

CD36 levels are decreased in cancer cell lines using retroviral orlentiviral vectors encoding miRNAs or shRNAs that target CD36. Knockdownefficiency is tested using qPCR analysis. Total RNA is extracted usingthe PicoPure RNA extraction kit (Arcturus) following the manufacturer'sprotocol. RNA is converted to cDNA using gScript™ cDNA supermix (Quantabiosciences). qPCR is performed with primers and iQ™ SYBER Green mastermix (Biorad, Hercule, Calif.). A standard protocol of initial denaturingat 95° C. for 10 min, 40 cycles of 95° C. for 10 sec, 60° C. for 30 sec,and 72° C. for 30 sec, followed by final extension at 72° C. for 5 minand melt curve analysis is applied on a BioRad CFX96 Real Time System(BioRad) coupled with Bio-Rad-CFX Manager software. The relativeabundance of each transcript compared with the control is calculatedutilizing the delta-Ct method.

In Vitro Cell Proliferation Assays

Cell proliferation is measured using the MTT(3-{4,5-Dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide,Sigma-Aldrich) assay. Cells are seeded in 50 uL growth medium in 96 wellculture plates and allowed to attach overnight. 50 uL of growth mediumplus two-fold concentrated treatment reagents were then added. Aftereach treatment time point, 10 uL of 5% MTT solution (buffered in PBS)was added to each well. Plates were incubated for an additional 4 h at37° C. to allow MTT to be metabolically converted into formazan crystalsat cell mitochondria. The formazan crystals were finally solubilized byadding 100 ul of 10% Sodium Dodecyl Sulphate in 50%N—N-Dimethylformamide to each microplate well. Absorbances at 550 and680 nm (corresponding to formazan salt and reference wavelengths,respectively) were measured using a colorimetry microplate reader. Wellscontaining only complete medium were used as controls. Each experimentwas performed twice, using six replicates for each drug concentration.

Metastasis Assay, Bioluminescence Imaging and Analysis

For experimental metastasis, 7-week old C57BL/6 mice are injected viatail vein with 1×10⁵ luciferase-labeled LLC cells. For orthotopic breastcancer cell injections, 5×10⁶ MDA-MB-231 or its metastatic variantMDA-MB-LM2 cells, are injected into CB-17 SCID mice fat pads in a volumeof 0.1 ml. Tumor growth and pulmonary metastases (following resection ofprimary tumor) were monitored by live animal bioluminescence imaging(Xenogen) once per week. For orthotopic prostate cancer cell injections,2×106 viable LN4 or cells were injected into the prostate gland of mice.

For in vivo determination of the metastatic burden, mice wereanaesthetized and injected intraperitoneally with 75 mg/kg ofD-luciferin (100 uL of 30 mg/mL in PBS). Metastatic growth was monitoredover time using bioluminescence imaging performed with mice in a supineposition 5 min after D-luciferin injection with a Xenogen IVIS systemcoupled to Living Image acquisition and analysis software (Xenogen). ForBLI plots, photon flux was calculated for each mouse by using the samecircular region of interest encompassing the thorax of the mouse.

Psap Peptide Administration

8-week old mice are treated with a Psap peptide (such as DWLPK (SEQ IDNO: 2), DWLP (SEQ ID NO: 3), or a modified version thereof), diluted inPBS, at a dose of 30 mg/kg/day via intraperitoneal injection for up totwo weeks.

Results

CD36 levels are measured in tissue samples from human subjects withcancer.

CD36 levels are knocked down in cancer cell lines and these cell lineswith reduced CD36 are injected into mice. Mice are then administered aPsap peptide. Tumor growth and metastatic burden are monitored. It isexpected that knockdown of CD36 in cancer cells will reduce theanti-cancer activity of a Psap peptide in vivo.

Example 3 Methods

Except were stated otherwise, the methods used in Example 3 are the sameas the methods used in Examples 1 and 2. The cell lines tested for CD36expression were pancreatic (AsPC1), ovarian (DF-14 and ID-8), breast(MDA-MB231 and LM2), prostate (PC3, PC3-M-LN4, LN-CAP, and LN-CAP-LN3),melanoma (B16-B16), and lung cancer (LLC) cell lines. All of these celllines are known in the art and/or commercially available.

Ovarian cancer cells expressing CD36 were treated with either control orthrombospondin (Tsp-1, either 100 ng, 500 ng or 1000 ng) and the percentof viable cells was measured at 0 hours or 48 hours.

For the ovarian cancer mouse model, 1 million ovarian cancer cellsexpressing luciferase were injected intraperitoneally. Treatment wasinitiated 17 days later with cisplatin (4 mg/kg QOD), the psap peptidedW1P (SEQ ID NO: 47, 40 mg/kg QD), a combination of cisplatin and psappeptide, or PBS QD. Luciferase intensity was measured on several daysbeginning on about day 17 and measured over time.

For the pancreatic cancer mouse model, 1×10⁶ AsPc1 human pancreaticcells were injected into the pancreas of SCID mice. Mice were treatedeither with a control or with the psap peptide dW1P (SEQ ID NO: 47, 20mg/kg/day or 40/mg/kg/day). Treatment began on day 25 and continueddaily for 21 days. The mice were then euthanized and the primary tumormass was measured. The presence or absence of ascites was also measured.

For the melanoma mouse model, B16-B16 cells were injected into mice.Mice were treated with either psap peptide dW1P (SEQ ID NO: 47, 10 or 40mg/kg) or control. The volume of the tumor was measured over time upuntil about 20-25 days post cell injection.

Results

Multiple cancer cell lines were tested for expression of CD36. It wasfound that CD36 protein was detectable in all cell lines tested, withparticularly high levels of CD36 detectable in AsPC1, DF-14, MDA-MB231,and PC3 cell lines (FIG. 5).

It was shown that ovarian cells expressing CD36 were sensitive to Tsp-1mediated cell killing in a dose-dependent manner (FIG. 6).

Two “high” CD36 cell lines (ovarian cancer cells and AsPC pancreaticcancer cells) and one “low” CD36 cell line (B16-B6 cancer cells) wereinjected into mice to study the effects of psap peptides on tumor growthand metastasis. It was found that the “high” CD36 cancer modelsregressed in response to psap peptide treatment (FIGS. 5 and 7). Theovarian cancer model also showed regression of metastatic disease (FIG.5). The pancreatic cancer model also showed inhibition of metastasisbecause only 1 of 19 mice treated with a psap peptide form ascites,while 4 of 10 mice treated with the control formed ascites. In the “low”CD36 melanoma model, it was found that treatment with a psap peptideinhibited primary tumor growth but did not cause tumor regression (FIG.8). These results show that “high” CD36 cancers are more likely tostrongly respond to psap peptide treatment (e.g., regression of primarytumor and/or metastasis), while “low” CD36 cancers are more likely tohave a weaker response (e.g., inhibition of primary tumor rather thanregression).

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present disclosure toits fullest extent. The specific embodiments are, therefore, to beconstrued as merely illustrative, and not limitative of the remainder ofthe disclosure in any way whatsoever. All publications cited herein areincorporated by reference for the purposes or subject matter referencedherein.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present disclosure, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the disclosure to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

What is claimed is:
 1. A method for evaluating a subject'sresponsiveness to treatment with a Psap peptide, the method comprising:determining a level of CD36 in a sample obtained from a subject havingcancer, wherein an elevated level of CD36 in the sample compared to acontrol level indicates that the subject is responsive to or likely tobe responsive to treatment with a Psap peptide.
 2. The method of claim1, wherein the level of CD36 in the sample is determined by performingan assay.
 3. The method of claim 1, wherein the method furthercomprises: identifying the subject with an elevated level of CD36 in thesample compared to the control level as responsive to or likely to beresponsive to treatment with a Psap peptide.
 4. The method of claim 3,wherein the method further comprises: administering to the subjectidentified as responsive to or likely to be responsive to treatment witha Psap peptide an effective amount of a Psap peptide to treat thecancer. 5-33. (canceled)
 34. A method for treating cancer in a subjectin need thereof, the method comprising: administering to the subject aneffective amount of a prosaposin (Psap) peptide, wherein the cancer ischaracterized by an elevated level of CD36 as compared to a controllevel of CD36, and wherein the Psap peptide is 4 to 9 amino acids inlength and comprises an amino acid sequence selected from the groupconsisting of CDWLPK (SEQ ID NO: 1), DWLPK (SEQ ID NO: 2), DWLP (SEQ IDNO: 3), and amino acid substitution variants thereof, wherein the aminoacid substitution variant comprises an amino acid substitution selectedfrom the group consisting of: a) Tyrosine (Y) for Tryptophan (W); b) anamino acid substitution for Leucine (L) selected from Valine (V),Alanine (A) or Glycine (G), or a non-canonical amino acid of similarsize, or a derivative thereof; c) Arginine (R) for Lysine (K); d) aD-isomer of Aspartic Acid (D) for an L-isomer of Aspartic Acid (D)and/or a D-isomer of Leucine (L) for a L-isomer of Leucine (L); and e) aD-isomer of Tryptophan (W) for an L-isomer of Tryptophan (W) and/or aD-isomer of Praline (P) for an L-isomer of Praline (P).
 35. The methodof claim 34, wherein the control level is a level of CD36 from anon-cancerous cell or tissue obtained from the subject having thecancer.
 36. The method of claim 34, wherein the control level is a levelof CD36 in a cell or tissue obtained from a healthy subject or apopulation of healthy subjects.
 37. The method of claim 34, wherein thecontrol level is a predetermined level.
 38. The method of claim 34,wherein the level of CD36 is a CD36 protein level.
 39. The method ofclaim 34, wherein the cancer is prostate cancer, breast cancer, ovariancancer, lung cancer, leukemia, pancreatic cancer, glioblastomamultiforme, astrocytoma, or melanoma.
 40. The method of claim 34,wherein the Psap peptide is a cyclic peptide.
 41. The method of claim34, wherein the non-canonical amino acid of similar size ismethylvaline, methylleucine, or sarcosine.
 42. The method of claim 34,wherein the control level is a level of CD36 from a non-cancerous cellor tissue obtained from the subject having the cancer.
 43. The method ofclaim 34, wherein the Psap peptide comprises one or more modificationsselected from the group consisting of acetylation, pegylation, andconjugation to a polymer that enhances serum half-life of the peptide.44. The method of claim 34, wherein the Psap peptide is a dimer, trimer,or multimer.
 45. The method of claim 34, wherein the Psap peptide is 6or fewer amino acids in length.